基因编辑技术在产溶剂梭菌中的应用

Application of gene-editing technology in solventogenic clostridia

产溶剂梭菌是一类能够利用碳源产生生物燃料的菌种,在可再生生物能源发展中具有巨大的应用潜力,但是目前针对这类菌的遗传操作工具相对匮乏,是其应用于工业生产的阻碍;在分子遗传学快速发展的背景之下,基因编辑技术成为研究微生物代谢调控机制以及遗传改造的首选工具.本文首先从来源组成、基本原理以及工作机制等方面分别介绍应用于产溶剂梭菌中不同类型的基因编辑技术,指出各自的优缺点;接着提出基因编辑技术在产溶剂梭菌中的3大应用前提:(1)掌握基因组序列信息,(2)突破自身限制性酶修饰系统的屏障,(3)建立外源DNA转化方法;进一步按照时间脉络综述基因编辑技术在产溶剂梭菌中的发展以及编辑工具的不断优化过程,描述其从随机失活到精准靶向失活、从低的转化效率到高效基因编辑的技术进步.针对CRISPR目前存在的Cas蛋白毒性问题、HDR的重组效率以及脱靶效应等技术难题,指出未来在产溶剂梭菌中应尝试探索攻克这些难题的方向,对产溶剂梭菌进一步的遗传改造,从而实现更高的工业应用价值.

Solventogenic clostridia are bacteria that can use carbon sources to produce biofuels,and they have great potential for application in renewable bioenergy.However,few manipulation tools for these bacteria have been developed in recent years,thus hindering their application in industrial production.With the rapid development of molecular genetics,gene-editing technology has become the preferred tool for studying microbial metabolism,regulation mechanisms,and genetic modification.We first introduce various types of gene-editing technologies applied to solventogenic clostridia in terms of source composition,basic principles,and working mechanisms and we highlight their respective advantages and disadvantages.In addition,three prerequisites for the application of gene-editing technology in solventogenic clostridia are proposed:(1)master genome sequence information,(2)breaking the barriers of its own restriction-modification system,and(3)establishment of a method for exogenous DNA transformation.We then review the application of gene-editing technology and the continuous optimization process of editing tools in solventogenic clostridia according to the time context.We describe the technological progress achieved from random inactivation to precisely targeted inactivation and from low transformation efficiency to efficient gene editing.Finally,we propose the current Cas protein toxicity problems,the recombination efficiency of HDR,and off-target effects in CRISPR,highlighting the broad application prospects of CRISPR technology in solventogenic clostridia and the directions that can be attempted in the future,looking forward to furthering genetic modification of solventogenic clostridia to achieve higher industrial application value.